Team Publications Controlling Dynamics and Function with the code

Year of publication 2009

Dorota Wloga, Danielle M Webster, Krzysztof Rogowski, Marie-Hélène Bré, Nicolette Levilliers, Maria Jerka-Dziadosz, Carsten Janke, Scott T Dougan, Jacek Gaertig (2009 Jun 15) TTLL3 Is a tubulin ligase that regulates the assembly of cilia. Developmental cell : 867-76 : DOI : 10.1016/j.devcel.2009.04.008

Summary

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer’s vesicle and olfactory placode. We also show that, in vivo, and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal and acts either directly or indirectly by inhibiting tubulin glutamylation.

Krzysztof Rogowski, François Juge, Juliette van Dijk, Dorota Wloga, Jean-Marc Strub, Nicolette Levilliers, Daniel Thomas, Marie-Hélène Bré, Alain Van Dorsselaer, Jacek Gaertig, Carsten Janke (2009 Jun 12) Evolutionary divergence of enzymatic mechanisms for posttranslational . Cell : 1076-87 : DOI : 10.1016/j.cell.2009.05.020

Summary

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

Christoph Maas, Dorthe Belgardt, Han Kyu Lee, Frank F Heisler, Corinna Lappe-Siefke, Maria M Magiera, Juliette van Dijk, Torben J Hausrat, Carsten Janke, Matthias Kneussel (2009 May 26)

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1 Team Publications Controlling Microtubule Dynamics and Function with the tubulin code

Synaptic activation modifies microtubules underlying transport of postsynaptic cargo. Proceedings of the National Academy of Sciences of the United States of America : 8731-6 : DOI : 10.1073/pnas.0812391106

Summary

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

Year of publication 2008

Peter Bieling, Stefanie Kandels-Lewis, Ivo A Telley, Juliette van Dijk, Carsten Janke, Thomas Surrey (2008 Dec 29) CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. The Journal of cell biology : 1223-33 : DOI : 10.1083/jcb.200809190

Summary

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end-binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated alpha-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2 Team Publications Controlling Microtubule Dynamics and Function with the tubulin code

Carsten Janke, Krzysztof Rogowski, Juliette van Dijk (2008 Jul 1) Polyglutamylation: a fine-regulator of protein function? ‘Protein Modifications: beyond the usual suspects’ review series. EMBO reports : 636-41 : DOI : 10.1038/embor.2008.114

Summary

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post- translational modification could be a potential regulator of diverse cellular processes.

Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, Bernard Edde, Carsten Janke (2008 Feb 15) Polyglutamylation is a post-translational modification with a broad range of substrates. The Journal of biological chemistry : 3915-22 : DOI : 10.1074/jbc.M705813200

Summary

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on , and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3 Team Publications Controlling Microtubule Dynamics and Function with the tubulin code

Year of publication 2007

Juliette van Dijk, Krzysztof Rogowski, Julie Miro, Benjamin Lacroix, Bernard Eddé, Carsten Janke (2007 May 11) A targeted multienzyme mechanism for selective microtubule polyglutamylation. Molecular cell : 437-48 : DOI : 10.1016/j.molcel.2007.04.012

Summary

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

Koji Ikegami, Robb L Heier, Midori Taruishi, Hiroshi Takagi, Masahiro Mukai, Shuichi Shimma, Shu Taira, Ken Hatanaka, Nobuhiro Morone, Ikuko Yao, Patrick K Campbell, Shigeki Yuasa, Carsten Janke, Grant R Macgregor, Mitsutoshi Setou (2007 Feb 27) Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function. Proceedings of the National Academy of Sciences of the United States of America : 3213-8 : DOI : 10.1073/pnas.0611547104

Summary

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular “traffic sign” for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild- type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 4 Team Publications Controlling Microtubule Dynamics and Function with the tubulin code

subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

Year of publication 2005

Carsten Janke, Krzysztof Rogowski, Dorota Wloga, Catherine Regnard, Andrey V Kajava, Jean- Marc Strub, Nevzat Temurak, Juliette van Dijk, Dominique Boucher, Alain van Dorsselaer, Swati Suryavanshi, Jacek Gaertig, Bernard Eddé (2005 Jun 17) Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science (New York, N.Y.) : 1758-62 : DOI : 10.1126/science.1113010

Summary

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin ligase- like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Year of publication 2004

Carsten Janke, Maria M Magiera, Nicole Rathfelder, Christof Taxis, Simone Reber, Hiromi Maekawa, Alexandra Moreno-Borchart, Georg Doenges, Etienne Schwob, Elmar Schiebel, Michael Knop (2004 Aug 5) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast (Chichester, England) : 947-62 : DOI : 10.1002/yea.1142

Summary

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 5 Team Publications Controlling Microtubule Dynamics and Function with the tubulin code

homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

Year of publication 2003

Catherine Regnard, Didier Fesquet, Carsten Janke, Dominique Boucher, Elisabeth Desbruyéres, Annette Koulakoff, Christine Insina, Pierre Travo, Bernard Eddé (2003 Oct 15) Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase. Journal of cell science : 4181-90 : DOI : 10.1242/jcs.00743

Summary

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360×103) displayed at least three major polypeptides of 32, 50 and 80×103, present in stochiometric amounts. We show that the 32×103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6